mmg_233_2013_genetics_genomicswikiaorg-20200214-history
Gentically Modified Wheat Plants
Introduction Salinity stress in agricultural crops is a serious factor in limiting productivity via the detrimental effects of excess sodium (Na+) on biological processes2 along with osmotic stress caused by a deficiency in water. One mechanism that has evolved universally in plants to acclimate to unfavorable environments is the accumulation of organic metabolites of low molecular weight, known collectively as compatible solutes3. Although metabolites comprising compatible solutes vary among plants, the major role is sustained in that they serve as organic osmolytes that increase the ability of cells within the plant to retain water naturally1. Greenhouse-grown Triticum aestivium wheat plants were genetically modified via transformation with the plasmid pBI-P5CS4 containing the genetic material required to synthesize proline, a compatible solute with strong potential. Specifically, an agrobacterium-mediated gene transfer through an indirect pollen system5 was used in the genetic modification. Screening of the wheat grains and southern, northern and western blot analyses were performed to ensure proper transfer along with a salt tolerance test and proline analysis to monitor the effect of salt-stress conditions on plant growth1. Genetic Modifications The plasmid pBI-P5CS contains the structural gene encoding kanamycin sulphate resistance, neomycin phosphotransferase-II (npt II) along with a reporter gene encoding β-glucuronidase (gus) and Vigna aconitifolia ''P5CS cDNA. Cells suspensions from an overnight culture of ''Agrobacterium ''strain LBA4404 tansformed with the pBI-P5CS plasmid5 were pipetted into each spikelets of a wheat ear while the spikelets in the middle of the ear were just before anthesis1. To screen for kanamycin resistance, the wheat grains were surface-sterilized and allowed to swell for at least 2 hours. The grains were then screened via a 2-step procedure that allowed for the elimination of infected grains. In this 2-step process, the grains were first screen on an agar medium with supplemented kanamycin. Following 4 days of growth, the best plantlets were transferred into glass jars with a reduced kanamycin content, that after 5 days allowed only certain surviving plants. These plants were transferred to soil and grown to full. Southern, northern and western blot analyses were then performed followed by a salt tolerance test in which identical two-week old transgenic and control wheat seedlings were grown in a greenhouse and were given increasing concentrations of sodium in a diluted solution over a 24-day watering treatment. The salt-stress conditions were monitored to observe the effects of proline, a compatible solute. A proline analysis was also done in which leaf tissue from both plants, transgenic and control, were frozen and ground in liquid nitrogen and, following centrifugation, used to determine proline concentration6 from the supernatant solution1. Goals The goal of the study was to demonstrate the heritable enhancement of salt tolerance within transgenic wheat plants through the genetic manipulation of the ability to synthesize proline1. Accomplishing this goal would suggest the possibility of increasing salt tolerance by genetic manipulations of proline biosyenthesis, allowing nonaccumulators or low-level accumulators to accumulate proline a protective levels1. It was found in the study that the production of proline seems to improve the salinity tolerance of transgenic wheat plants along with the potential use of these transgenic plants for agricultural use in saline soils1. Although it is thought that salt tolerance is a complex trait and thus multiple traits are responsible for it, this study showed that the overexpression of a single gene encoding proline can improve the salinity tolerance of the wheat plant, ''Triticum aestivium. References 1Sawahel W A, Hassan A H. Generation of transgenic wheat plants producing high levels of the osmoprotectant proline. Biotechnology Letters. 2002; 24:721-25. 2Zhang H, Blumwald E. Transgenic salt-tolerant tomato plants accumulate salt in foliage but not in fruit. Nat. Biotechnol. 2001; 19:765-68. 3Sakamoto A, Murata N. Genetic engineering of glycinebetaine synthesis in plants: current status and implications for enhancement of stress tolerance. J. Exp. Bot. 2000; 51:81-88. 4Kavi Kishor B, Hong Z, Miao G, Hu C, Verma D. Overexpression of 1-pyrroline-5-carboxylate synthetase increases proline production and confers osmotolerance in transgenic plants. Plant Physiol. 1995; 108:1387-94. 5Hess D, Dressler K, Nimmrichter R. Transformation experiments by pipetting Agrobacterium into the spikelets of wheat (Triticum aestivum ''L.). ''Plant Sci. 1990; 72:233-44. 6Bates L, Waldren R, Teare I. Rapid determination of free proline for water-stress studies. Plant Soil. 1973; 39:205-7.